Rotaviruses are the single most important etiologic agent of infectious gastroenteritis (diarrhea), which is the leading cause of infant death in the world.
Of the estimated 5 to 10 million infant deaths caused by acute infectious gastroenteritis yearly [Walsh et al, New Engl. J. Med., 301:967 (1979)], rotaviruses cause between 10 and 40% of the total deaths [deZoysa and Feachem, Bull. WHO, 63:569 (1985)]. Rotavirus-induced infectious gastroenteritis is one of the ten leading causes of infant death, even in developed nations [Ho et al, 27th Interscience Conf. Antimicrobiol Agents Chemotherapy, p2 (1987)].
Rotaviruses of primate and bovine origin are spherical viruses, about 70 nm in diameter and characterized by a double capsid structure. The rotavirus genome has eleven segments of doublestranded RNA and an RNA polymerase. Each segment of RNA is a gene that codes for a single protein gene product. Rotavirus gene segment numbering is arbitrary, conventionally based on molecular size and electrophoretic migration.
A majority of the currently identified animal and human rotaviruses are designated as Group A rotaviruses, and share common cross-reactive antigens [Estes et al, Immunochemistry of Viruses, Elsevier, Amsterdam:389 (1984)]. Different species of rotaviruses are distinguishable by distinct serotype-specific virus surface antigens, which are most easily detected in conventional serum-neutralization (SN) tests. In a SN test, an antiserum prepared against a purified virus of a specific serotype scores a higher SN titer with a virus of a homologous serotype than with a virus of a heterologous serotype.
Among human rotaviruses, at least six serotypes are now recognized-serotypes 1, 2, 3, 4, 8 and 9 [Wyatt et al, Infect. Immunol., 37:110 (1983); Matsuno et al, J. Virol., 54:623 (1985); and Clark et al, J. Clin. Microbiol., 25:1757 (1987)], with serotypes 1 through 4 causing the majority of human infections.
Currently available knowledge on the cross-immunity of various serotypes in animals and humans has provided contradictory results with regard to the necessity for serotype specific antigenic stimulation to provide immune protection against rotavirus infection. See, for example, several reports on vaccine challenge studies in animals [Wyatt et al, Science, 203:548 (1979); Zissis et al, J. Infect. Dis., 148:1061 (1983); Sheridan et al, J. Infect. Dis., 149:434 (1984)]. See, also, several reports on rotavirus vaccines evaluated in animals and humans [Mebus et al, J. A. V. M. A., 163:880 (1973); Thurber et al, Canad. Vet. J., 17:197 (1976); Vesikari et al, Lancet, 2:807 (1983); De Mol et al, Lancet, 2:108 (1986); Clark et al, Amer. J. Dis. Children, 140:350 (1986); Kapikian et al, Vaccines, New York, Cold Spring Harbor Lab., 357 (1985); Losonsky et al, Pediatr. Infect. Dis., 5:25 (1986); and Santosham et al, 27th Int. Conf. Antimicrobiol. Agents and Chemotherapy, p. 99 (1987)].
Efforts to develop an effective rotavirus vaccine have proven disappointing to date H F. Clark, "Rotavirus Vaccines," Vaccines, Plotkin, S. A., Mortimer, E. A., eds. (Philadelphia, W. B. Saunders: 1988), p. 517; H F. Clark, Immunization Monitor, 3:3 (1989); R. S. Daum et al, "New Developments in Vaccines," Adv Pediatr Infect Dis., 6:1 (1991). See, also, "WHO News and Activities," Bull. WHO, 67(5): 583-587 (1989), reporting on the efficacy of rotavirus vaccines.
Animal-origin vaccines have often been unsatisfactory when evaluated in clinical trials involving orally administered living virus. For example, although the immunization of infants with bovine rotavirus vaccine strain RIT4237 has proven safe and induced measurable serum antibody response in 60-80% of recipients, early protective responses detected in trials in Finland were followed by unsuccessful clinical trials in Africa and in the United States [see, Daum et al, cited above].
Another candidate tested for use as a human vaccine, simian-origin rotavirus RRV (MMU 18006), was found to be more immunogenic than RIT4237 but also provided inconsistent protection against rotavirus disease. [G. A. Losonsky et al, Pediatr. Infect. Dis., 5:25 (1986); and T. Vesikari et al, J. Infect. Dis., 153:832 (1986)]. A bovine-origin rotavirus of low-tissue culture passage level, strain WC3, which is safe in infants and causes a serum antibody response in 70-95% of recipients, also provides inconsistent and limited protection.
Human rotaviruses identified to date are much more difficult to cultivate in cell culture than rotaviruses of animal origin. Human origin rotaviruses have previously been isolated successfully in cell culture using methods which were first described in K. Sato et al, Arch. Virol., 69:155 (1981) and T. Urasawa et al., Microbiol. Immunol., 25:1025 (1981). These methods involved inoculation of infected stool suspensions into roller tube cultures of cells of the African green monkey cell line MA104, followed by several "blind" passages of the inoculated cells. Such viruses have usually grown poorly in comparison with other species' rotaviruses; i.e., to titers 10-100 fold less than those often obtained with certain bovine and simian origin rotaviruses. Thus, in general, cell culture-adapted human rotaviruses are generally known to replicate inefficiently in vitro. This lack of an efficient means to produce larger quantities of human virus in vitro also hampers research into the virus and its antigenic components.
To date, human rotavirus isolates have been propagated only in a few cell types of simian origin. Rotaviruses of human origin have not been propagated in human diploid cell strains (HDCS). HDCS are currently considered the ideal cell substrate for production of virus vaccines for use in humans [See, e.g., M. R. Hilleman, J. Med. Virol., 31:5 (1990)].
Additionally, the potential pathogenicity of human rotavirus isolates is largely unknown. In view of these above-mentioned difficulties, human rotaviruses have not been avidly pursued for vaccine use.
The failure of animal-origin vaccines to provide consistent protection and the inability to obtain readily cultivatable human rotaviruses for vaccine use have led to interest in the formation of rotavirus reassortants as potential vaccine candidates. A reassortant is formed by incorporating genes from one rotavirus coding for desired antigens (i.e., type-specific antigens) into another rotavirus. This incorporation, called "reassortment", is possible because of the segmented nature of the RNA genome of a rotavirus and its high frequency of gene reassortment during coinfection of distinct parental rotaviruses.
In rotavirus reassortants of bovine/simian origins, it was observed that the v.p.7 and v.p.4 major outer capsid proteins were independently capable of inducing a protective immune response in mice against virulent rotavirus challenge when administered at high dosages [Offit et al, J. Virol., 60(2):491-496 (1986); Offit et al, J. Virol., 57(2):376-378 (1986)].
Previously known reassortant rotaviruses proposed for use as human vaccine candidates involved the replacement of a single gene product encoding the v.p.7 antigen of an animal rotavirus. The 38 kd v.p.7 antigen of gene 9 or 8 of the animal virus has been replaced with the v.p.7 encoding gene of a human serotype rotavirus. U.S. Pat. No. 4,571,385 describes a method of producing a rotavirus reassortant from certain human and animal parental strains by combining the human rotavirus with a cultivatable animal rotavirus and selecting for desired reassortants with an antibody specific for the 34-38 kd glycoprotein, v.p.7 of the animal virus. [See, also, Midthun et al, J. Virol., 53:949 (1985); Midthun et al, J. Clin. Microbiol., 24:822 (1986).]
Compared to animal rotavirus vaccine candidates, the human/animal reassortants have been found to elicit an increased, but disappointing, incidence of human serotype-specific responses. Additionally, RRV reassortant derivatives have induced undesirable side effects, e.g., fever and gastrointestinal symptoms, in a significant proportion of vaccinees in clinical trials [Vesikari et al, cited above; and Losonsky et al, cited above].
Reassortants have been formed by co-infection of two human strains, but to date they have never been tested for vaccine use. [See, e.g., Urasawa et al., J. Gen. Virol., 67:1551-1559 (1987)].
Additional difficulties encountered in inducing a vigorous and effective antibody response to rotavirus after oral administration of a potential vaccine to infants are significant interference with an active immune response in infants possessing pre-existing antibody of maternal derivation (virtually universal in very young infants) and difficulty in obtaining a universal "booster" antibody response with a second oral dose [Clark et al, Vaccine, 8:327 (1990)]. Additionally, the ability of a reassortant to induce neutralizing antisera is not itself indicative of the usefulness of the reassortant as an effective and safe vaccine for humans.
There is currently great interest in pursuing the possibility that these obstacles may be overcome by administering a killed rotavirus vaccine parenterally (e.g., by intramuscular or subcutaneous inoculation). In the case of an inoculated vaccine, it is especially important that the virus be propagated in an acceptable cell culture substrate, of which the most desirable is any human diploid cell strain. Prior to the present invention neither a human nor animal rotavirus has been reported to replicate in HDCS.
Therefore, there remains a need in the art for human origin rotavirus and reassortants thereof capable of growing to high titer in cell culture, vaccines containing these viruses or reassortants, and diagnostic and research reagents in the field of rotavirus research.